Search

From superficial brown discolorations on the caps and stalks of deformed sporophores of Agaricus bisporus, bacteria were consistently isolated. On the basis of biochemical, nutritional and pathogenic characteristics, both on tissue blocks and on whole sporophores of A. bisporus, the above bacteria were identified as strains of Pseudomonas agarici.

The biological activities of the lipodepsipeptides (LDP) white line-inducing principle (WLIP), produced by Pseudomonas reactansNCPPB1311, and tolaasin I, produced by P. tolaasii NCPPB2192, were compared. Antimicrobial assays showed that both LDP inhibited the growth of fungi—including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.—chromista, and gram-positive bacteria. Assays of the two LDP on blocks of Agaricus bisporus showed their capacity to alter the mushrooms' pseudo-tissues though WLIP was less active than that of tolaasin I. Contrary to previous studies, tolaasin I was found to inhibit the growth of gram-negative bacteria belonging to the genera Escherichia, Erwinia, Agrobacterium, Pseudomonas, and Xanthomonas. The only gram-negative bacterium affected by WLIP was Erwinia carotovora subsp. carotovora. Both WLIP and tolaasin I caused red blood cell lysis through a colloid-osmotic shock mediated by transmembrane pores; however, the haemolytic activity of WLIP was greater than that of tolaasin I. Transmembrane pores, at a concentration corresponding to 1.5 × C50, showed a radius between 1.5 and 1.7 ± 0.1 nm for WLIP and 2.1 ± 0.1 nm for tolaasin I. The antifungal activity of WLIP together with the finding that avirulent morphological variants of P. reactans lack WLIP production suggests that WLIP may play an important role in the interaction of the producing bacterium P. reactans and cultivated mushrooms.

Fluorescent pseudomonads isolated from different lesions on caps and/or stipes of cultivated Pleurotus ostreatus were identified as strains of Pseudomonas tolaasii or showed the White Line Assay (WLA) feature of P. ‘reactans’ or were WLA-negative fluorescent Pseudomonas spp. Pseudomonas tolaasii was consistently associated with brown-reddish blotches on P. ostreatus pseudo-tissues, and in the pathogenicity assays caused depressed dark brown lesions with deliquescence on Agaricus bisporus pseudo-tissues blocks and brown-reddish blotches and yellow discoloration on P. ostreatus sporocarps. Pseudomonas ‘reactans’ and the WLA-negative fluorescent Pseudomonas spp. were mostly associated with superficial yellow lesions on P. ostreatus sporocarps, and in pathogenicity assays caused light or dark brown discoloration, depending on the isolates, on A. bisporus pseudo-tissues blocks and the yellow discoloration of P. ostreatus sporocarps. The results of this study indicate that the aetiology of lesions on cultivated P. ostreatus involves a complex composed of interactions between P. tolaasii, P. ‘reactans’ and Pseudomonas spp., but that individually these bacteria cause different symptoms. This is the first report where the pathogenicity features of these pathogens has been clearly ascertained, and that has fully satisfied Koch’s postulates for the bacteria on the host mushroom. On the basis of virulence, biochemical and physiological characters, the isolates of P. ‘reactans’ and Pseudomonas spp. responsible for yellowing of oyster mushroom belong to several species of Pseudomonas.

Cultivars of the Fagioli di Sarconi are a pool of high value common bean (Phaseolus vulgaris L.) traditional cultivars selected from various landraces. These cultivars are protected by the European Union (Reg. CEE n° 1263/96) with the mark PGI (Protected Geographical Indication) and are cultivated in Basilicata (southern Italy) for the production of dry seeds. Fagioli di Sarconi cultivars are susceptible to common bacterial blight (CBB), a disease caused by the varieties fuscans and non-fuscans of Xanthomonas axonopodis pv. phaseoli. Five Fagioli di Sarconi cultivars (Tondino bianco, Verdolino, Cannellino, Tabacchino and Ciuoto) were artificially inoculated with two virulent strains of this bacterium, representative of 59 recently characterized strains of both the fuscans and non-fuscans varieties, with the aim to evaluate the susceptibility of these cultivars to the pathogens. Four CBB–resistant breeding–lines were used for comparison. Suspensions of the bacterium were injected into the first trifoliate leaflets of bean plants and produced typical CBB symptoms on the Fagioli di Sarconi cultivars, and hypersensitive necrotic lesions on the CBB–resistant breeding–lines. When cultivars were inoculated with the strain of the variety fuscans, Tondino bianco, Verdolino and Cannellino were more susceptible than Tabacchino and Ciuoto, whereas when they were inoculated with the non–fuscans strains, Tondino bianco, Tabacchino and Verdolino were more susceptible than Ciuoto and Cannellino. The tolerant cultivars appeared good candidates for the introgression of CBB–resistance characters in a breeding programme. The varying response of the traditional Fagioli di Sarconi bean cultivars to artificial inoculation suggests that several plant and pathogen factors are involved in the interaction between X. a. pv. phaseoli varieties and the bean cultivars.

Pseudomonas tolaasii is the main bacterial pathogen of several mushroom species. In this paper we report that strains of P. tolaasii produce volatile substances inducing in vitro mycelia growth inhibition of Pleurotus ostreatus and P. eryngii, and Agaricus bisporus and P. ostreatus basidiome tissue blocks brown discoloration. P. tolaasii strains produced the volatile ammonia but not hydrogen cyanide. Among the volatiles detected by GC-MS, methanethiol, dimethyl disulfide (DMDS), and 1-undecene were identified. The latter, when assayed individually as pure compounds, led to similar effects noticed when P. tolaasii volatiles natural blend was used on mushrooms mycelia and basidiome tissue blocks. Furthermore, the natural volatile mixture resulted toxic toward lettuce and broccoli seedling growth. In contrast, pure volatiles showed different activity according to their nature and/or doses applied. Indeed, methanethiol resulted toxic at all the doses used, while DMDS toxicity was assessed till a quantity of 1.25 μg, below which it caused, together with 1-undecene (≥10 μg), broccoli growth increase.

Six rhizobacteria isolated from common bean and able to protect bean plants from the common bacterial blight (CBB) causal agent, were in vitro evaluated for their potential antifungal effects toward different plant pathogenic fungi, mostly soil-borne. By dual culture assays, the above bacteria resulted producing diffusible and volatile metabolites which inhibited the growth of the majority of the pathogens under study. In particular, the latter substances highly affected the mycelium growth of Sclerotinia sclerotiorum strains, one of which was selected for further studies either on mycelium or sclerotia. Gas chromatographic analysis of the bacterial volatiles led to the identification of an array of volatile organic compounds (VOCs). Time course studies showed the modification of the VOCs profile along a period of 5 days. In order to evaluate the single detected VOC effects on fungal growth, some of the pure compounds were tested on S. sclerotiorum mycelium and their minimal inhibitory quantities were determined. Similarly, the minimal inhibitory quantities on sclerotia germination were also defined. Moreover, observations by light and transmission electron microscopes highlighted hyphae cytoplasm granulation and ultrastructural alterations at cell organelles, mostly membranes, mitochondria, and endoplasmic reticulum. The membranes appeared one of the primary targets of bacterial volatiles, as confirmed by hemolytic activity observed for the majority of pure VOCs. However, of interest is the alteration observed on mitochondria as well.

Among the main (> or = 0.7%) components of some essential oils, considerable antibacterial activity was shown by terpenoid and phenylpropanoid derivatives containing phenol and alcohol functionalities. A reduced or no activity was shown by those derivatives containing ketones, aldehydes, ethers, and ester functionalities as well as the remaining terpenoids. Eugenol emulsion treatments (1-8 mg/mL) of bean seeds bearing about 2.6 x 10(6) cfu/seed of strain ICMP239 of Xanthomonas campestris pv. phaseoli var. fuscans determined a highly significant reduction of the bacteria on seeds. In particular, eugenol at 4 mg/mL disinfect seeds bearing about 7.0 x 10(2) cfu/seed and lower densities. However, after 72 h, incubation treatments with 2, 4, and 8 mg/mL of eugenol caused germination reduction of 3%, 7%, and 16%, respectively, which was significantly different from the controls. No effect on germination was observed with 1 mg/mL eugenol emulsion treatment. These data indicate eugenol as potentially useful for bean seed disinfection from X. campestris pv. phaseoli var. fuscans. Further studies on the effects on seed vitality and on formulation of essential oils are needed.

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.

The biological activities of the lipodepsipeptides (LDP) white line-inducing principle (WLIP), produced by Pseudomonas reactans NCPPB1311, and tolaasin I, produced by R tolaasii NCPPB2192, were compared. Antimicrobial assays showed that both LDP inhibited the growth of fungi-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.--chromista, and gram-positive bacteria. Assays of the two LDP on blocks of Agaricus bisporus showed their capacity to alter the mushrooms' pseudo-tissues though WLIP was less active than that of tolaasin I. Contrary to previous studies, tolaasin I was found to inhibit the growth of gram-negative bacteria belonging to the genera Escherichia, Erwinia, Agrobacterium, Pseudomonas, and Xanthomonas. The only gram-negative bacterium affected by WLIP was Erwinia carotovora subsp. carotovora. Both WLIP and tolaasin I caused red blood cell lysis through a colloid-osmotic shock mediated by transmembrane pores; however, the haemolytic activity of WLIP was greater than that of tolaasin I. Transmembrane pores, at a concentration corresponding to 1.5 x C50, showed a radius between 1.5 and 1.7 +/- 0.1 nm for WLIP and 2.1 +/- 0.1 nm for tolaasin I. The antifungal activity of WLIP together with the finding that avirulent morphological variants of P. reactans lack WLIP production suggests that WLIP may play an important role in the interaction of the producing bacterium P. reactans and cultivated mushrooms.

Essential oils extracted by hydrodistillation from fruits of Cuminum cyminum L. and Carum carvi L. were analyzed by gas chromatography (GC) and GC-mass spectrometry (MS). The main components of C. cyminum oil were p-mentha-1,4-dien-7-al, cumin aldehyde, gamma-terpinene, and beta-pinene, while those of the C. carvi oil were carvone, limonene, germacrene D, and trans-dihydrocarvone. Antibacterial activity, determined with the agar diffusion method, was observed against Gram-positive and Gram-negative bacterial species in this study. The activity was particularly high against the genera Clavibacter, Curtobacterium, Rhodococcus, Erwinia, Xanthomonas, Ralstonia, and Agrobacterium, which are responsible for plant or cultivated mushroom diseases worldwide. In general, a lower activity was observed against bacteria belonging to the genus Pseudomonas. These results suggest the potential use of the above essential oils for the control of bacterial diseases.

Essential oils were extracted from the fruits of Coriandrum sativum L. and Foeniculum vulgare Miller var. vulgare (Miller) and assayed in vitro for antibacterial activity to Escherichia coli and Bacillus megaterium, bacteria routinely used for comparison in the antimicrobial assays, and 27 phytopathogenic bacterial species and two mycopathogenic ones responsible for cultivated mushroom diseases. A significant antibacterial activity, as determined with the agar diffusion method, was shown by C. sativum essential oil whereas a much reduced effect was observed for F. vulgare var. vulgare oil. C. sativum and F. vulgare var. vulgare essential oils may be useful natural bactericides for the control of bacterial diseases of plants and for seed treatment, in particular, in organic agriculture. The significant antibacterial activity of essential oils to the bacterial pathogens of mushrooms appears promising.

Pseudomonas tolaasii, the causal organism of brown blotch disease of Agaricus bisporus and of the yellowing of Pleurotus ostreatus, was shown to produce in culture tolaasin I (1), tolaasin II (2), and five other minor metabolites, tolaasins A, B, C, D, and E (3-7). These compounds were demonstrated to be important in the development of the disease symptoms. This paper reports on the structural elucidation, based essentially on NMR studies and MS spectra, and biological activity of the above lipodepsipeptides (3-7). All the above analogues showed differences in the peptide moiety, as observed in other lipodepsipeptides of bacterial origin, and maintained the beta-hydroxyoctanoyl phi chain at the N-terminus, except tolaasin A, in which the acyl moiety was a gamma-carboxybutanoyl phi moiety. Among the target microorganisms used (fungi, yeast, and bacteria) the Gram-positive bacteria were the most sensitive, although the antimicrobial activity appeared to be correlated to the structural modification in the different analogues. The structure-activity relationships of these toxins are discussed.

The 18-amino acid cytolytic lipodepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii, is the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D-amino acids in its N-terminal region, then alternates L- and D-amino acids, and bears a C-terminal lactone macrocycle composed of 5-residues. The solution structure of tolaasin in sodium dodecyl sulfate was studied by 2D-NMR spectroscopy and molecular dynamics simulated annealing calculations. Tolaasin forms an amphipathic left-handed alpha-helix in the regionDPro2-DalloThr14 comprising the sequence of seven D-amino acids and the adjacent L-D-L-D-D-region. To the best of our knowledge, this is the first recognized example of a left-handed alpha-helix including both D- and L-amino acids. The lactone macrocycle adopts a "boat-like" conformation and is shifted from the helical axis as to form a "golf-club" overall conformation. These structural features will be of importance in understanding, and preventing, tolaasin's role in the bacterial colonization of the host plant, and its toxic action on cells. Furthermore, the observed antimicrobial activity together with the potential resistance to enzymatic degradation and the increased antigenicity (both due to the presence of L- and D-amino acids) strongly suggests for tolaasin a potential role as a template model for the design of new therapeutic antibacterial molecules., (Copyright 2003 Wiley-Liss, Inc.)

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position:

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP(22)Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP(22)Phv did not differ substantially from the previously characterized syringopeptin 22 (SP(22)) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium.

The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR). The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)-3-hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH). A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3'. The potential biological meaning of this peculiar lipid A is also discussed.

An O-specific polysaccharide containing 2-acetamidino-2-deoxy-beta-D-glucopyranose (Glcp2Am), 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAc4NAc, bacillosamine) and 2,4-di-(N-acetyl-L-alanylamino)-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAlaAc4NAlaAc) was isolated from the phenol-soluble lipopolysaccharide fraction of the mushroom-associated bacterium Pseudomonas reactans. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a linear trisaccharide-repeating unit, as shown below:-->3)-beta-D-QuipNAlaAc4NAlaAc-(1-->3)-alpha-D-Glcp2Am-(1-->3)-alpha-D-QuipNAc4NAc(1-->To our knowledge, this is the first complete O-chain structure reported for the lipopolysaccharide of a mushroom-associated bacterium.